In the treatment of various ischemia-related diseases associated with arteriosclerosis, the revascularization treatment by local or transvenous transplantation of autologous myeloblasts (mononuclear cells) has been provided as an advanced medical technique. Under current situation, it is thought that in most cases fractions containing hematopoietic stem cells such as CD34-positive cells and CD133-positive cells have anti-ischemia effects (see Patent Literature 1), and therefore the improvement of therapeutic performance by purification of cells with these surface antigens is anticipated. Though bone marrow and umbilical cord blood may become a source of such cells, as they contain relatively many undifferentiated cells, peripheral blood contains very few CD34-positive cells and CD133-positive cells. Therefore, when peripheral blood is used, large scale mobilization of bone marrow progenitor cells by granulocyte colony-stimulating factor (G-CSF) etc. is commonly practiced, and still, collecting enough number of cells required for a certain therapeutic effect can be difficult.
As for patients with pre-existing diseases such as arteriosclerosis or diabetes and elderly people, in addition to the difficulty in collecting bone marrow, a concern exists about declines of the cellular function itself (see Non Patent Literatures 1 and 2). As a new approach, a method using ex vivo amplification of progenitor cells contained in umbilical cord blood has been reported (see Patent Literature 2). Furthermore, the utilization of embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) is anticipated as a future source of such undifferentiated cells (stem cells). However, there is still a long way to the realization thereof.
There is a report suggesting the use of endothelial progenitor cells (EPCs) obtained by inducing differentiation of mononuclear cells contained in bone marrow or peripheral blood (see Non Patent Literature 3), in contrast to methods using rare (hematopoietic) stem cells as a source in the revascularization treatment. In this report, mononuclear cells are cultured in a medium, such as EBM2, that contains cytokines including vascular endothelial growth factor (VEGF) and that is optimized for culturing vascular endothelial cells, and the cells collected as adherent cells or floating cells are called EPCs.
Adherent cells obtained by inducing differentiation of murine peripheral blood or bone marrow mononuclear cells in EGM2-MV medium supplemented with 10% FBS, in a culture dish treated with rat vitronectin uptake acetylated low-density lipoprotein (acetylated LDL) and show affinity for lectin. Among the adherent cells, cells in fusiform shape and cells in round shape are mixed. The proportion of the former is higher within the first week of culturing, while with longer culturing cells in pavement shape grow densely, which are deduced to be derived from the latter. Thus, the adherent cells obtained by the method described above include distinct cell populations, it is reasonable to consider that EPCs are included in the latter fraction. However, the occurrence of subculturable cells from pavement-shaped colonization is very low. Fusiform cells seen abundant in the initial stage of the differentiation-inducing culture swell and extend, and decline substantially in their survival rate.
Transplanting (local and systemic administration) cells obtained by culturing mouse mononuclear cells for a short period of about 1 week into an small animal, such as mouse, that suffers from myocardial or lower limb ischemia is known to lead to recovering from ischemia. Namely, it is known that (a population of) cells obtained by inducing differentiation of mononuclear cells have an effect to promote vascularization. On the other hand, it is also known that cells (cell populations) obtained by inducing differentiation of mononuclear cells have a reduction effect of the hypoxia region of tumor (cancer) tissue (see Patent Literature 3) and liver fibrosis suppression effects (see Patent Literature 4). However, these cells do not necessarily express the antigens called EPC markers (see Non Patent Literature 4), such as CD34 and VEGF receptor 2 (VEGFR2/Flk-1/KDR) on the cell membrane. It is not clear whether these cells have lost the stem cell antigens during the differentiation process of hematopoietic stem cells, or they are of a population of cells derived from non-hematopoietic stem cells that do not express the stem cell antigens from the beginning.
Similarly, it has been reported that in a culture period of about 1 week, adherent cells obtained by differentiation-inducing culture of human peripheral blood mononuclear cells in EGM2-MV medium supplemented with 10% FBS, in a culture dish treated with human fibronectin keep round-to-fusiform shapes well, have the ability to promote lumen formation of human vascular endothelial cells such as HUVEC (see Non Patent Literature 5), and suppress necrosis associated with lower limb ischemia in a nude mouse (see Non Patent Literature 6). Also reported are clinical trial results showing that autotransplantation of human peripheral blood mononuclear cells treated with similar differentiation induction improved cardiac function after myocardial infarction, and transplanted cells are defined as EPCs expressing surface antigens such as Flk-1, CD31, CD105, VE-cadherin (see Non Patent Literature 7). However, because the culture period was as short as 3 days, amplification of hematopoietic stem cells in large quantities is reasonably deduced to be rather unlikely, and the possibility that they were cells derived from monocyte line is undeniable.
With long-term culture with inducing differentiation of mononuclear cells, whether cells of interest will be efficiently obtained, or whether cellular functions and the quality of the cells will be kept well is not clear yet. Although hypoxia environments are known to keep EPCs in undifferentiated states (see Non Patent Literature 8), and to have the antioxidant effect (see Non Patent Literature 9) during the induction of differentiation. Because EGM2-MV media containing FBS were used in most of reports so far, they are not directly applicable to clinical applications (administration to human).
The induction of differentiation of mononuclear cells as a source into EPCs having the ability to differentiate into vascular endothelial cells is anticipated as an alternative method of the revascularization treatment with CD34 or CD113-positive hematopoietic stem cells. However, as noted above, (a population of) adherent cells obtained by inducing differentiation of mononuclear cells are of a heterogenous population(s). A clinically applicable technique for efficiently obtaining only EPCs suitable for revascularization treatment by differentiation induction has not been established yet.
CD11b is one of the hemocyte differentiation antigens that are expressed mainly in monocytes and lymphocytes. CD11b-positive cells include some of cells that are responsible for immunological surveillance and lymphocytes, such as macrophages, dendritic cells, natural killer cells (NK cells). On the other hand, it is also known that in abnormal new blood vessels seen in cancer or like, the expression of a receptor for an angiogenic factor, such as VE-cadherin, VEGF receptor 1 (VEGFR1), SDF-1 receptor (CXCR4), and angiopoietin-1 receptor (Tie-2), together with CD11b antigen is found. Thus, the possibility that non-stem cells expressing these markers may have an important role in vascularization, for example, via differentiating into cells having a vascularization promoting effect is suggested (see Non Patent Literature 10).
On the other hand, CD11b-positive cells include also relatively undifferentiated fractions. It is reported that a part of CD11b-positive cells in bone marrow can differentiate into CD31 antigen-positive vascular endothelial cells or smooth muscle actin (SMA) antigen-positive parietal cells in the presence of an angiogenic factor, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) or the like (see Non Patent Literature 11). In addition, relatively young cells, such as vascular progenitor cells (VPC) obtained by inducing differentiation of embryonic stem cells (see Non Patent Literature 12) are thought to have properties similar to these, and their ultimate direction of differentiation may depend on the environment.
The facts described above suggest the possibility that some CD11b-positive cells directly become neovascular components, or they are indirectly involved in the promotion of angiogenesis or the stabilization of new blood vessels through cytokine production. However, a differentiation induction system that is free from samples derived from animal, such as fetal bovine serum (FBS, FCS), for inducing differentiation into cells with the functions such as revascularization, angioplasty, blood vessel stabilization, using, as a source, non-hematopoietic stem cells expressing monocyte differentiation markers such as CD11b, which are relatively abundant in mononuclear cells has not yet established. Moreover, cells that express monocyte markers such as CD11b may differentiate into endothelial cells in tumor vessels (see Non Patent Literature 13), and also may locate around blood vessels without differentiating into vascular endothelial cells (for example, CD11b-positive cells coexpressing Tie2; see Non Patent Literature 14); and practically it is difficult to distinguish their characteristics and roles strictly in a body with tumor (cancer) (see Non Patent Literature 15). Thus, it is not clear whether cells obtained by inducing differentiation of CD11b expression mononuclear cells function as EPCs in the end.